Francisella tularensis is the etiologic agent of tularemia. This gram negative bacterium is a potential agent of bioterrorism because of its high mortality, extremely low infective dose, ability to survive for long periods of time outside of an animal host, and numerous transmission mechanisms including inhalation, ingestion, skin contact, mucous membrane contact, and insect vectors. Little is known about the mechanisms of F. tularensis pathogenesis and host resistance. F. tularensis will survive within macrophages, however macrophage-like cells infected with these organisms become apoptotic within 24 hours following inoculation. The first aim of this proposal is to examine the mechanism(s) by which F. tularensis provoke host cell death in macrophages. F. tularensis transposon insertion mutants that fail to induce macrophage apoptosis will be isolated and the mutations characterized to define bacterial products responsible for promoting host cell death. These mutant strains will be tested for potential attenuation in a mouse model of tularemia. The mechanism of apoptosis induction by F. tularensis will be determined using mouse gene chips possessing known pro- and anti- apoptotic gene sequences to compare macrophage mRNA levels from control and F. tularensis infected cells. The second aim is to isolate Francisella specific CD4 T cell hybrids and determine the differences in the epitopes recognized in C57Bl/6 mice infected with live or heat killed bacteria. E. coli clones expressing the apoptosis inducing genes identified in the first aim will be created and used to isolate specific CD4 T cell hybrids. The ultimate aim will be to create T cell clones of defined cytokine secretion (Th1 or Th2) (by using the defined peptide epitopes) and determine the effect of the clones on pathogenesis and protection. These aims are directed towards examining the molecular basis for F. tularensis pathogenesis, combined with an approach to elucidate potential immune response targets that could lead to improved prophylaxis against tularemia.